There is intense interest in the circuits that guide stem cell behavior. Due to the difficulty in identifying stem cells in tissues, most work has centered on intrinsically-acting factors that control stem cells, or on identifying culture conditions that allow their expansion while yet maintaining their undifferentiated state. Thus, there is significantly less known about the microenvironment that comprises a stem cell's natural niche, even though it provides many of the signals that govern fundamental stem cell properties. Understanding niche-stem cell interactions is central to unraveling the circuitry necessary to use these cells in regenerative medicine. One of the most well-understood stem cell-niche systems is the Drosophila testis, because the stem cells and their niche are precisely defined and the outlines of a regulatory program are in place. The testis niche maintains both germline and somatic stem cells (GSCs and SSCs, respectively). Here, two essential SSC factors, lines and zfh-1, are focused upon, and their study has led us to a reconsideration of the rules governing this well-established stem cell-niche model. This proposal addresses conceptually significant facets of stem cell-niche biology. First, there are precious few systems where one can at high resolution investigate the genetic circuitry that discriminates a stem cell from its niche cell during development. Aim 1A seeks to define the role of lines and its partner protein bowl in mediating SSC-niche fate choice in gonadogenesis. Since a neural stem cell can also generate cells of its niche, interest in the circuitry identified here will be high. Second, Hedgehog signaling has been implicated in the maintenance of various stem cell types, including cancer stem cells, but the particular characteristics that Hh signaling assigns to stem cells have remained elusive. Aim 1B seeks to define the role of Hh in SSCs, in particular, assessing which of the defining characteristics of stem cells are regulated by Hh signaling. Third, understanding the mechanisms that repress differentiation in stem cells is a major goal of much work in regenerative medicine. Aim 2A seeks to identify the mechanism whereby Zfh-1 blocks differentiation and confers stem cell properties to somatic cells. Aim 2A seeks to define how Zfh-1 cooperates (non-autonomously) with STAT activation in GSCs. Aim 2C proposes complementary approaches to identify genes under Zfh-1 control that help accomplish both of these tasks. The prospects are also excellent that our work can contribute in a fundamental manner to this aspect of stem cell biology. Public Health Relevance: There is intense interest in the circuits that guide stem cell behavior. Understanding niche-stem cell interactions is central to unraveling the circuitry necessary to use these cells in regenerative medicine. This proposal addresses conceptually significant facets of stem cell-niche biology.